Tuberculosis (TB) is a major global health problem, and new intervention strategies are urgently needed to reduce its deadly impact. The long term objective of this project is to better understand how Mycobacterium tuberculosis (Mtb) responds to its host environment at the gene regulatory level so that effective strategies can be developed to prevent TB disease. Gene regulation is a critical aspect of Mtb's ability to cause infection, but little is known about gene regulatory mechanisms in Mtb during either active or latent infection. Previous studies have established that a set of acr-coregulated genes (ACGs) is coordinately expressed in response to hypoxia, shallow standing growth conditions, and within macrophages. ACG expression has been associated with Mtb virulence and establishment of latent TB infection, suggesting an important role for these genes during TB pathogenesis. However, neither the regulation nor function of these genes is understood. A multidisciplinary approach will be used to address the mechanisms of ACG regulation and function in Mtb at the molecular, genetic and cellular levels, with a particular focus on the effects of latency-associated environmental conditions. Specific aims include: Aim 1: Characterization of specific mechanisms of DosR-dependent ACG regulation at the molecular and biochemical levels, using promoter:reporter fusions, mutagenesis and DNA-protein binding assays. Studies will focus on the roles of DosR and ACG motif binding sites in regulation of devR, Rv3130c and Rv2623; Aim 2: Characterization, using genetic and biochemical approaches, of a new level of DosR-independent negative regulation associated with ACG 5'UTRs, including Rv2623. The cis and trans factors associated with this 5'UTR repression will be identified, and the biological roles of the 5'UTR repressor and Rv2623 regulation will be assessed using in vitro and in vivo models; Aim 3: Evaluation of a slow growth Mtb culture model compared with existing latency models in the context of new information on the importance of carbon dioxide to Mtb metabolism and its ability to abrogate hypoxia-associated growth arrest. Culture models will be compared to intramacrophage and in vivo conditions with regards to gene expression and lipid accumulation. Characterization of gene regulatory mechanisms in Mtb, and the environmental cues to which tubercle bacilli respond, will contribute to our understanding of the factors needed for the establishment of TB infection, and may identify new targets for TB vaccines, therapeutics, and/or diagnostic purposes.